CN110441445B - Method for constructing cockscomb HPLC characteristic fingerprint spectrum and measuring flavonoid component content - Google Patents
Method for constructing cockscomb HPLC characteristic fingerprint spectrum and measuring flavonoid component content Download PDFInfo
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Abstract
The invention relates to a method for constructing a cockscomb HPLC characteristic fingerprint spectrum and flavonoid component content determination. The establishment method of the cockscomb medicinal material HPLC characteristic fingerprint comprises the following steps: (1) precisely weighing a cockscomb medicinal material, and preparing a cockscomb test sample solution; (2) analyzing the cockscomb sample solution by adopting a high performance liquid chromatograph to obtain a cockscomb HPLC characteristic fingerprint; the method for measuring the content of the flavonoid components comprises the following steps: (1) the method comprises the following steps of (1) sucking mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a high performance liquid chromatograph, drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking a peak area as a vertical coordinate, and determining a linear regression equation; (2) injecting the cockscomb pattern product solution to be detected into a high performance liquid chromatograph, and measuring a corresponding peak area; (3) calculating by a linear regression equation to obtain; the method has good precision, stability and reproducibility, and can better evaluate the quality of flos Celosiae Cristatae.
Description
Technical Field
The invention relates to the technical field of detection of traditional Chinese medicinal materials, and particularly discloses a method for constructing an HPLC (high performance liquid chromatography) characteristic fingerprint spectrum of cockscomb and measurement of flavonoid component content.
Background
The flos Celosiae Cristatae is dried inflorescence of flos Celosiae Cristatae (Celosia cristata L) of semen Celosiae of Amaranthaceae, and has effects of astringing to stop bleeding, stopping leukorrhagia, and relieving dysentery. Sweet and astringent in flavor, entering liver meridian and entering blood system. "compendium of materia Medica": cockscomb can treat haemorrhoids with bleeding, red and white diarrhea, metrorrhagia, red and white leucorrhea, and red and white leucorrhea. Cockscomb originates from tropical regions of Africa, America and Asia, the Tang generation is introduced into China, the Ming generation is planted generally, and the cockscomb is planted nationwide.
At present, quality research on cockscomb is only reported, most documents only carry out single fingerprint spectrum research on cockscomb, and do not carry out research on various components of cockscomb, so that great limitation exists, so far, a method for simultaneously measuring fingerprint spectrums capable of reflecting cockscomb medicinal material characteristic information and flavonoid components of cockscomb medicinal material by using only one chromatographic system does not exist, and the problem needs to be solved urgently. The traditional Chinese medicine components are complex, and the content of only a few compounds is not comprehensive as the evaluation index of quality. The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which can mark the chemical characteristics of a traditional Chinese medicine or a traditional Chinese medicine preparation by adopting a certain analysis means after the traditional Chinese medicine or the traditional Chinese medicine preparation is properly processed.
Disclosure of Invention
The invention aims to provide a method for constructing the HPLC characteristic fingerprint of cockscomb and a method for measuring the flavonoid component content thereof, aiming at the defects and shortcomings in the prior art.
The technical problem to be solved by the invention is realized by the following technical scheme:
a method for constructing an HPLC characteristic fingerprint of a cockscomb medicinal material comprises the following steps:
(1) precisely weighing a cockscomb medicinal material, and preparing a cockscomb test sample solution;
(2) analyzing the cockscomb sample solution by adopting a high performance liquid chromatograph to obtain the HPLC characteristic fingerprint of the cockscomb medicinal material.
As a preferred scheme, the chromatographic conditions analyzed by the high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 28-32 ℃; performing gradient elution by taking acetonitrile as a mobile phase A and taking a 0.18-0.22% phosphoric acid water solution as a mobile phase B; the flow rate is 0.9-1.1 ml/min; the detection wavelength is 254-365 nm; the sample injection amount is 8-15 mu l, and the phosphoric acid aqueous solution contains triethylamine with the volume fraction of 0.15-0.25%.
As a most preferred scheme, the chromatographic conditions analyzed by the high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 30 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using a 0.20% phosphoric acid aqueous solution as a mobile phase B; the flow rate is 1.0 ml/min; the detection wavelength is 290 nm; the sample amount is 10 mul, and the phosphoric acid aqueous solution contains triethylamine with the volume fraction of 0.20 percent.
As a preferred embodiment, the gradient elution conditions are: the volume fraction of acetonitrile is changed to 13-18% and the volume fraction of phosphoric acid is changed to 87-82% in 0-5 min; 5-6 min, wherein the volume fraction of acetonitrile is changed to 18-20%, and the volume fraction of phosphoric acid is changed to 82-80%; 6-18 min, wherein the volume fraction of acetonitrile is changed to 20%, and the volume fraction of phosphoric acid is changed to 80%; for 18-23 min, the volume fraction of acetonitrile is changed to 20-38%, and the volume fraction of phosphoric acid is changed to 80-62%; 23-32 min, wherein the volume fraction of acetonitrile is changed to 38%, and the volume fraction of phosphoric acid is changed to 62%; 32-38 min, wherein the volume fraction of acetonitrile is changed to 38-55%, and the volume fraction of phosphoric acid is changed to 62-45%; 38-43 min, the volume fraction of acetonitrile is changed to 55%, and the volume fraction of phosphoric acid is changed to 45%.
As a preferred scheme, the test solution is prepared by a method comprising the following steps: taking 0.08-0.12 g of cockscomb medicinal material powder, precisely adding 40-60 ml of mixed solution, heating for 55-65 minutes, cooling, weighing, complementing the weight loss by the mixed solution, shaking up, filtering, and taking the subsequent filtrate to obtain the cockscomb medicinal material powder.
The mixed solution as a preferable scheme is composed of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 45-65: 20-30: 15 to 25.
As a most preferred embodiment, the test solution is prepared by a method comprising the steps of: precisely weighing 0.1g of cockscomb medicinal powder, placing into a conical flask with a plug, precisely adding 50ml of mixed solution, weighing, refluxing in boiling water bath for 60 min, cooling, weighing again, supplementing the lost weight with the mixed solution, shaking up, filtering, and taking the subsequent filtrate.
As a most preferable scheme, the mixed solution is composed of ethanol, water and hydrochloric acid, and the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 50: 20: 15.
as a preferable scheme, the preparation method of the mixed reference substance solution comprises the step of adding an organic solvent to prepare the mixed reference substance solution containing 20-30 mu g of kaempferide, 1-3 mu g of isorhamnetin and 1-3 mu g of kaempferol-4 '-O-methyl ether in each 1ml of the mixed reference substance solution, so as to obtain the kaempferol-4' -O-methyl ether mixed reference substance solution.
As a most preferable scheme, the preparation method of the mixed reference substance solution comprises the step of adding methanol to prepare the mixed reference substance solution containing 25.4603 mu g of kaempferide, 2.05461 mu g of isorhamnetin and 2.0846 mu g of kaempferol-4' -O-methyl ether in each 1ml of the mixed reference substance solution, so as to obtain the mixed reference substance solution.
The invention also provides a method for measuring the content of flavonoid components in the cockscomb medicinal material, which comprises the following steps:
(1) the method comprises the following steps of (1) sucking mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a high performance liquid chromatograph, drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking a peak area as a vertical coordinate, and determining a linear regression equation;
(2) injecting the cockscomb pattern product solution to be detected into a high performance liquid chromatograph, measuring the corresponding peak area,
(3) and calculating by a linear regression equation to obtain the target.
Preferably, the flavonoid components are kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether.
As a preferred scheme, the chromatographic conditions analyzed by the high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 28-32 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution with the volume fraction of 0.18-0.22% as a mobile phase B; the flow rate is 0.9-1.1 ml/min; the detection wavelength is 300-400 nm; the sample injection amount is 8-15 mu l; the phosphoric acid aqueous solution contains triethylamine with the volume fraction of 0.15-0.25%.
As a most preferred scheme, the chromatographic conditions analyzed by the high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 30 ℃; performing gradient elution by taking acetonitrile as a mobile phase A and taking phosphoric acid aqueous solution with volume fraction of 0.2% as a mobile phase B; the flow rate is 1 ml/min; the detection wavelength is 365 nm; the sample amount is 10 mul; the phosphoric acid aqueous solution contains triethylamine with a volume fraction of 0.2%.
As a preferred embodiment, the gradient elution conditions are: 0-15 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%; for 15-20 min, the volume fraction of the mobile phase A is changed to 35-63%, and the volume fraction of the mobile phase B is changed to 65-37%; and 20-28 min, wherein the volume fraction of the mobile phase A is 63%, and the volume fraction of the mobile phase B is 37%.
As a preferable scheme, the cockscomb sample solution to be tested is prepared by a method comprising the following steps: taking 0.18-0.22 g of cockscomb medicinal powder to be detected, precisely adding 40-60 ml of mixed solution, heating for 55-65 minutes, cooling, weighing, complementing the weight loss by the mixed solution, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
The mixed solution as a preferable scheme is composed of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 45-65: 20-30: 15 to 25.
As a most preferred scheme, the cockscomb sample solution to be tested is prepared by a method comprising the following steps: precisely weighing 0.2g of cockscomb medicinal powder, placing into a conical flask with a plug, precisely adding 50ml of mixed solution, weighing, refluxing in boiling water bath for 60 min, cooling, weighing again, supplementing the lost weight with the mixed solution, shaking up, filtering, and taking the subsequent filtrate.
The mixed solution is composed of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 50: 20: 15.
as a preferable scheme, the preparation method of the mixed reference substance solution comprises the step of adding an organic solvent to prepare the mixed reference substance solution containing 15-25 mu g of kaempferide, 1-3 mu g of isorhamnetin and 1-3 mu g of kaempferol-4 '-O-methyl ether in each 1ml of the mixed reference substance solution, so as to obtain the kaempferol-4' -O-methyl ether mixed reference substance solution.
As a most preferable scheme, the preparation method of the mixed reference substance solution is that methanol is added to prepare the mixed reference substance solution containing 20 mu g of kaempferide, 2 mu g of isorhamnetin and 2 mu g of kaempferol-4' -O-methyl ether in each 1ml, and the mixed reference substance solution is obtained.
Has the advantages that: (1) the invention firstly constructs the HPLC characteristic fingerprint spectrum of the cockscomb medicinal material and the determination method of the flavonoid component thereof; (2) the characteristic fingerprint spectrum of the cockscomb medicinal material constructed by the invention fully shows the chemical component characteristics of the cockscomb medicinal material; (3) the characteristic fingerprint constructed by the invention comprehensively reflects the characteristic peak information of the sample, and the method is stable, high in precision and better in reproducibility; (4) the method for measuring the flavonoid components in the cockscomb medicinal material provided by the invention can measure the content of three flavonoids in the cockscomb medicinal material more quickly, has higher accuracy, and can better evaluate the quality of the cockscomb medicinal material.
Drawings
FIG. 1 is an overlay of HPLC fingerprints of 15 batches of cockscomb medicinal materials.
FIG. 2 is a peak chart of HPLC fingerprint spectra of 15 batches of cockscomb medicinal materials, wherein peak 8 is kaempferide, peak 9 is isorhamnetin, and peak 12 is kaempferol-4' -0-methyl ether.
FIG. 3 is a chromatogram peak identification chart of a cockscomb drug as a reference, wherein A is a reference solution; b is a test solution; 1: kaempferide; 2: isorhamnetin; 3: kaempferol-4' -0-methyl ether.
FIG. 4 is a graph of cluster analysis results of cockscomb sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Construction of cockscomb medicinal material characteristic fingerprint
The main apparatus is as follows: waters ARC high performance liquid chromatograph (Waters corporation, usa); waters HSS T3 column (4.6 mm. times.250 mm, 5 μm); model one ten thousandth balance ME204E (mettler-toledo, usa); model XP26 millionths of a day (mettler-toledo, usa); Milli-Q Integral 5 ultra pure water machine (Merck Millipore, Germany); FW100 pulverizer for Chinese herbal medicines (Tensted instruments, Tianjin);
the cockscomb medicinal materials are taken from different Chinese medicinal material markets and drugstores in all the countries.
TABLE 1 cockscomb medicinal material source
The main reagents are as follows: kaempferol (kaempferol) (batch No. 110861) 201611; source: China institute for testing and drug, content: 95.5%); isorhamnetin (batch No. 110860-201611; source: China institute for food and drug assay; content: 99.8%); kaempferol-4' -O-methyl ether (batch number: 491-54-3; source: Shanghai Shidande biological Co., Ltd.; content: 98%); ethanol (Fuyu fine chemical Co., Tianjin) was analyzed; the liquid phase is prepared from phosphoric acid (Mimi European chemical reagent Co., Tianjin, Kemi), triethylamine (Mimi European chemical reagent Co., Tianjin, Denk) and acetonitrile (Merck) by chromatographic grade, and water is ultrapure water (prepared by laboratories).
Test method
Preparation of cockscomb medicinal material sample
Precisely weighing 0.1g of cockscomb medicinal powder, placing into a conical flask with a plug, precisely adding 50ml of mixed solution, weighing, refluxing in boiling water bath for 60 min, cooling, weighing again, supplementing the lost weight with the mixed solution, shaking up, filtering, and taking the subsequent filtrate.
The mixed solution consists of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 50: 20: 15.
mixed control solutions
Precisely weighing appropriate amount of kaempferol reference substance, isorhamnetin reference substance, and kaempferol-4 '-O-methyl ether reference substance, precisely weighing, and adding methanol to obtain mixed reference substance solution containing 25.4603 μ g of kaempferol, 2.05461 μ g of isorhamnetin, and 2.0846 μ g of kaempferol-4' -O-methyl ether per 1 ml.
Chromatographic conditions
Using a Waters HSS T3 chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 mu m; the column temperature is 30 ℃; performing gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.20% phosphoric acid aqueous solution as a mobile phase B; the flow rate is 1.0 ml/min; the detection wavelength is 290 nm; the amount of sample was 10. mu.l. The phosphoric acid aqueous solution contains 0.20% triethylamine.
TABLE 2 gradient elution flow match ratio
Data processing
And (3) processing and analyzing HPLC data of the cockscomb medicinal material sold in the market by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software and SPSS 20.0.
Establishment of HPLC fingerprint
Precision test
And (3) continuously feeding sample solution for 6 times, recording a chromatogram, taking kaempferide as a reference peak, calculating that the RSD of the relative retention time of each common peak is less than 3.0%, and the RSD of the relative peak area is less than 3.0%, thereby indicating that the precision of the instrument is good.
Stability test
Sampling the test solution for 0, 2, 4, 8, 12, 18 and 24 hours respectively, recording a chromatogram, taking kaempferide as a reference peak, calculating that the RSD of each common peak relative retention time is less than 3.0 percent, and the RSD of the relative peak area is less than 3.0 percent, wherein the result shows that the test solution is stable within 24 hours.
Repeatability test
Establishment of cockscomb medicinal material fingerprint
Determination of common peaks
Taking 15 batches of cockscomb drug samples, preparing a sample solution according to a sample solution preparation method, carrying out sample injection measurement, carrying out common peak identification on 15 batches of cockscomb drug fingerprints by using traditional Chinese medicine chromatography fingerprint similarity evaluation software, and generating a reference map. The results are shown in FIGS. 1 and 2. The retention time and peak area of the common peak for each batch of samples are shown in Table 3, with peak 8 as a reference. All common peaks are stable, have fingerprint characteristics, and can be preliminarily determined as index component groups of cockscomb medicinal materials. By contrast with the reference, peak No. 8 is kaempferol peak, peak No. 9 is isorhamnetin peak, and peak No. 12 is kaempferol-4' -0-methyl ether peak, as shown in FIG. 3. The kaempferide peak is selected as a reference peak due to good separation effect and stable peak appearance.
TABLE 3 common peak retention time and peak area of cockscomb medicinal material fingerprint
Evaluation of similarity of fingerprint spectra of medicinal materials in each production area
The common mode (R) generated by 15 batches of cockscomb medicinal materials is used as a comparison fingerprint, the similarity range of the 15 batches of cockscomb medicinal materials and the comparison fingerprint is 0.859-0.988, except that the similarity of S5 and S15 and the comparison fingerprint is less than 0.9, the similarity of other batches of medicinal materials and the comparison fingerprint is more than 0.9, and the result shows that other production places medicinal materials show good similarity. The similarity of cockscomb medicinal materials from different sources is relatively close, which indicates that the quality of the cockscomb sold in the market is stable.
TABLE 4 cockscomb sample fingerprint similarity evaluation
Sample cluster analysis of cockscomb medicinal material
Quantifying the area of each chromatographic peak relative to the sample weighing, performing systematic clustering on 15 batches of cockscomb drug samples by using SPSS20.0 software, and adopting an interclass average number coupling method and an included angle cosine as a distance formula of sample similarity. Clustering analysis divides the cockscomb medicinal material samples into 4 types, wherein the type I comprises S1, S3, S7, S8, S9 and S10, namely the northern river sample, Guangxi and a batch of Jiangsu samples are clustered into one type; the class II comprises S2, S4 and S6, namely, a batch of samples in Hebei, Anhui and Jiangsu are gathered into a class; group III includes S11, S12, S13 and S14, i.e. 4 samples of Guangdong province are grouped into one group; the IV group comprises S15, and the V group comprises S5, namely a batch of Jiangsu samples and a batch of Guangdong samples are gathered into one group. In general, the quality of cockscomb medicinal materials sold in Guangdong and Guangxi provinces is similar, but samples from different sources can be gathered into one group, which indicates that the quality difference of the samples from different sources is not large and is consistent with the evaluation result of similarity analysis. The specific results are shown in FIG. 4.
Example 2
Measuring the content of flavonoid components in the cockscomb medicinal materials by the following steps:
(1) sucking mixed reference substance solutions with different concentrations and injecting the mixed reference substance solutions into a high performance liquid chromatograph to obtain an HPLC characteristic fingerprint spectrum;
(2) injecting the cockscomb pattern product solution to be detected into a high performance liquid chromatograph, measuring a corresponding peak area, drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and the peak area as a vertical coordinate, and determining a linear regression equation;
(3) and calculating by a linear regression equation to obtain the target.
Preparation of cockscomb sample solution to be detected
Precisely weighing 0.2g of cockscomb pattern product powder to be detected, placing the cockscomb pattern product powder into a conical flask with a plug, precisely adding 50ml of mixed solution, weighing, refluxing in boiling water bath for 60 minutes, cooling, weighing again, complementing the lost weight with the mixed solution, shaking up, filtering, and taking the subsequent filtrate to obtain the cockscomb pattern product.
The mixed solution consists of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 50: 20: 15.
mixed control solutions
Precisely weighing appropriate amount of kaempferide reference substance, isorhamnetin reference substance, and kaempferol-4 '-O-methyl ether reference substance, precisely weighing, and adding methanol to obtain mixed reference substance solution containing kaempferide 20 μ g, isorhamnetin 2 μ g, and kaempferol-4' -O-methyl ether 2 μ g per 1 ml.
Chromatographic conditions
Using a Waters HSS T3 chromatographic column with the specification of 4.6mm multiplied by 250mm and 5 mu m; performing gradient elution at the column temperature of 30 ℃, using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.20% phosphoric acid solution (containing 0.2% triethylamine) as a mobile phase B; the flow rate is 1.0 ml/min; the detection wavelength is 365 nm; the amount of sample was 10. mu.l.
TABLE 5 gradient elution mobile phase ratio
Precisely weighing 4.188mg of kaempferide reference substance, placing in a 10ml measuring flask, and adding methanol to scale to obtain the final product with concentration of 509.206 μ g/mg. Precisely weighing isorhamnetin reference substance 2.196mg, placing into a 10ml measuring flask, and adding methanol to scale to obtain the final product with concentration of 219.1608 μ g/mg. Precisely weighing 2.431mg of kaempferol-4' -O-methyl ether reference substance, placing in a 10ml measuring flask, and adding methanol to scale to obtain concentration of 238.238 μ g/mg.
Precisely sucking 8ml of the kaempferide reference substance stock solution, 1.5ml of the isorhamnetin reference substance stock solution and 1.4ml of the kaempferol-4 '-O-methyl ether reference substance stock solution into a 20ml measuring flask, and adding methanol to prepare a reference substance linear mother solution containing 203.6824 mu g of kaempferide, 16.4371 mu g of isorhamnetin and 16.6767 mu g of kaempferol-4' -O-methyl ether per 1 ml. Respectively and precisely sucking linear mother liquor 0.25ml, 0.5ml, 1.25ml, 2.5ml and 5.0ml to 10ml volumetric flasks to prepare mixed reference solution with kaempferide concentrations of 5.0921, 10.1841, 25.4603, 50.9206 and 101.8412 mu g/ml, isorhamnetin concentrations of 0.4109, 0.8219, 2.0546, 4.1093, 8.2185 and 16.4371 mu g/ml and kaempferol-4' -O-methyl ether concentrations of 0.4169, 0.8338, 2.0846, 4.1692, 8.3383 and 16.6767 mu g/ml, making a standard curve, injecting the standard curve into a liquid chromatograph, and drawing the standard curve by taking the sample injection concentration as a horizontal coordinate and the peak area as a vertical coordinate.
TABLE 6 Linear regression equation and Linear Range
Sample application recovery test
Taking 0.1g of cockscomb medicinal material powder with the determined content, precisely weighing, weighing 9 parts in parallel, adding a reference substance with the sample content of 1.5:1:0.5 into each part, preparing a test solution according to the preparation method of the test solution, and preparing 9 parts of the test solution. Each aliquot was measured by pipetting 10. mu.l precisely and the sample recovery was calculated.
TABLE 7 sample recovery test results (n = 9)
Sample assay
Taking 15 batches of cockscomb medicinal materials with different sources, preparing a sample solution according to a sample solution preparation method, carrying out sample injection measurement, and calculating the content results of kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether, wherein the content results are shown in a table 8. The result shows that the highest value of the total content of kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether in 15 batches of samples is 11.34, the lowest value is 4.327mg/g, the content difference is not large, and the result is consistent with the result of a fingerprint.
TABLE 8 measurement results of flavonoid component content in cockscomb medicinal material
At present, the research on the fingerprint of cockscomb medicinal materials is only reported, and the quality of cockscomb medicinal materials cannot be scientifically evaluated. The experiment researches the flavonoid components in the cockscomb medicinal materials, establishes the fingerprint of the cockscomb medicinal materials for the first time, and comprehensively evaluates the flavonoid components of the cockscomb by combining the content determination method of kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether. The flavonoids in the cockscomb have the effects of promoting blood coagulation, resisting trichomonas vaginalis and enhancing immunity, and have the same effects of astringing and stopping bleeding, leucorrhea and dysentery as the cockscomb, and the experiments do not research the flavonoids in the cockscomb and need to be further researched. The research uses a fingerprint analysis method and takes the contents of kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether as indexes, so that the quality of the cockscomb medicinal materials sold in the market is comprehensively evaluated, and a foundation is laid for the quality control research of the cockscomb medicinal materials.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A method for constructing a cockscomb HPLC characteristic fingerprint spectrum is characterized by comprising the following steps:
(1) precisely weighing a cockscomb medicinal material, and preparing a cockscomb test sample solution;
(2) analyzing the cockscomb sample solution by adopting a high performance liquid chromatograph to obtain a cockscomb medicinal material HPLC characteristic fingerprint;
the chromatographic conditions of the high performance liquid chromatograph analysis are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 28-32 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution with the volume fraction of 0.18-0.22% as a mobile phase B; the flow rate is 0.9-1.1 ml/min; the detection wavelength is 254-365 nm; the sample injection amount is 8-15 mu l; the phosphoric acid aqueous solution contains triethylamine with the volume fraction of 0.15-0.25%;
the gradient elution conditions were: the volume fraction of the mobile phase A is changed to 13-18% and the volume fraction of the mobile phase B is changed to 87-82% in 0-5 min; 5-6 min, the volume fraction of the mobile phase A is changed to 18-20%, and the volume fraction of the mobile phase B is changed to 82-80%; 6-18 min, the volume fraction of the mobile phase A is changed to 20%, and the volume fraction of the mobile phase B is changed to 80%; for 18-23 min, the volume fraction of the mobile phase A is changed to 20-38%, and the volume fraction of the mobile phase B is changed to 80-62%; 23-32 min, changing the volume fraction of the mobile phase A to 38% and the volume fraction of the mobile phase B to 62%; 32-38 min, the volume fraction of the mobile phase A is changed to 38-55%, and the volume fraction of the mobile phase B is changed to 62-45%; and (3) 38-43 min, wherein the volume fraction of the mobile phase A is changed to 55%, and the volume fraction of the mobile phase B is changed to 45%.
2. The method for constructing the HPLC characteristic fingerprint of cockscomb according to claim 1, wherein the sample solution is prepared by a method comprising the following steps: taking 0.08-0.12 g of cockscomb medicinal material powder, precisely adding 40-60 ml of mixed solution, heating for 55-65 minutes, cooling, weighing, complementing the weight loss by the mixed solution, shaking up, filtering, and taking a subsequent filtrate to obtain the cockscomb medicinal material powder; the mixed solution consists of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is 45-65: 20-30: 15 to 25.
3. A method for measuring the content of flavonoid components in cockscomb medicinal materials is characterized by comprising the following steps:
(1) the method comprises the following steps of (1) sucking mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a high performance liquid chromatograph, drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking a peak area as a vertical coordinate, and determining a linear regression equation;
(2) injecting the cockscomb pattern product solution to be detected into a high performance liquid chromatograph, and measuring a corresponding peak area;
(3) calculating by a linear regression equation to obtain;
the chromatographic conditions of the high performance liquid chromatograph analysis are as follows: octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is 28-32 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution with the volume fraction of 0.18-0.22% as a mobile phase B; the flow rate is 0.9-1.1 ml/min; the detection wavelength is 300-400 nm; the sample injection amount is 8-15 mu l; the phosphoric acid aqueous solution contains triethylamine with the volume fraction of 0.15-0.25%;
the gradient elution conditions were: 0-15 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%; for 15-20 min, the volume fraction of the mobile phase A is changed to 35-63%, and the volume fraction of the mobile phase B is changed to 65-37%; 20-28 min, wherein the volume fraction of the mobile phase A is 63%, and the volume fraction of the mobile phase B is 37%;
the flavonoids comprise kaempferide, isorhamnetin and kaempferol-4' -O-methyl ether.
4. The method for measuring the content of flavonoid components in the cockscomb medicinal material according to claim 3, wherein the mixed reference solution is prepared by the following method: adding organic solvent to prepare a mixed reference solution containing 15-25 mu g of kaempferide, 1-3 mu g of isorhamnetin and 1-3 mu g of kaempferol-4 '-O-methyl ether in each 1ml of the solution, thus obtaining the kaempferol-4' -O-methyl ether.
5. The method for measuring the content of flavonoid components in the cockscomb sample medicinal material according to claim 3, wherein the cockscomb sample solution to be measured is prepared by a method comprising the following steps: taking 0.18-0.22 g of cockscomb medicinal powder to be detected, precisely adding 40-60 ml of mixed solution, heating for 55-65 minutes, cooling, weighing, complementing the weight loss by the mixed solution, shaking up, filtering, and taking a subsequent filtrate to obtain the cockscomb medicinal powder; the mixed solution consists of ethanol, water and hydrochloric acid, wherein the volume ratio of the ethanol to the water to the hydrochloric acid is as follows: 45-65: 20-30: 15 to 25.
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